2340 Wei Ye ∗∗ Hanxu Fu ∗∗ Lin Xie Lijun Zhou Tai Rao Qian Wang Yuhao Shao Jingcheng Xiao Dian Kang Guangji Wang ∗ Yan Liang Key Lab of Drug Metabolism & Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, China Received January 25, 2015 Revised March 26, 2015 Accepted April 4, 2015
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Research Article
Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid–liquid extraction with n-butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C18 column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one-step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ± 15) and precision (% RSD 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II. Keywords: Liquid chromatography / Pharmacokinetics / Tandem mass spectrometry / Ziyuglycoside I / Ziyuglycoside II DOI 10.1002/jssc.201500102
1 Introduction Chinese herbal medicines had been widely used to prevent and treat diseases for thousands of years, and they are becoming more and more popular for prescription in clinical therapy for their high efficacy, ease of access, low costs, and the belief of their better compatibility with the human body and few adverse effects [1, 2]. Sanguisorba officinalis L. is a natural plant that has been traditionally used for the treatment of inflammatory and metabolic diseases, including diarrhea, chronic intestinal infections, duodenal ulcers, bleeding, etc [3–6]. A variety of chemical constituents, including triterpenoid saponins, triterpenoid flavonoids, anthraquinones, steroids, were isolated from Sanguisorba officinalis L., and pharmacological studies on their anticancer, anti-inflammatory and antioxidant properties were reported in last decade years [7–9]. Ziyuglycoside I and ziyuglycoside II are the major effective ingredients of triterpenoid saponins exacted from Sanguisorba officinalis L., and many studies have focused on Correspondence: Dr. Liang, 24 Tongjia Lane, Nanjing, Jiangsu 210009, China E-mail: [email protected] Fax: +86 25 83271060.
Abbreviations: LLOQ, lower limit of quantification; SRM, selected-reaction monitoring C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
their pharmacological activities [10–12]. In 2008, Kim et al. reported that ziyuglycoside I isolated from the S. officinalis root extract had free radical scavenging activities and elastase inhibition activities, and could be used as an active ingredient in new anti-wrinkle cosmetic products [11]. Besides, ziyuglycoside II was reported can significantly inhibit the growth of MDA-MB-435 cells through blocking cell cycle progression as well as by inducing cell apoptosis [12]. In 2012, Zhang et al. developed a LC–MS method to simultaneously quantitative analysis of seven major triterpenoids in Pyrola decorata H. Andres. However, this method was not applicable to pharmacokinetic study due to diverse matrix and different validation standard [13]. To our best knowledge, no research about the pharmacokinetic study for ziyuglycosides was reported until now. In most cases, the lack of knowledge in biological disposition was one of the major obstacles to understanding whether the Chinese herbal medicines or single compound of western medicine, and pharmacokinetic study had been proved as a crucial step to uncover the pharmacologically active substances and identify drug targets [14, 15]. Herein, a novel quantitative method for ziyuglycoside I and ziyuglycoside II was developed based on a Shimadzu
∗ Additional corresponding author: Dr. Guangji Wang E-mail: [email protected] ∗∗ These two authors contributed equally to this work.
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LC–MS/MS 8050 system, and validated considering the specificity, linearity, matrix effects, precision, accuracy, and sensitivity in accordance to the criteria established by the US Food and Drug Administration. The applicability of the method was tested using rat plasma samples obtained after intragastric administration of Sanguisorba officinalis L. extract at a dosage of 2.0 g/kg.
2 Materials and methods 2.1 Materials Sanguisorba officinalis L. extract was purchased from Nanjing Qingze Medical Technological Development (Nanjing, China), the contents of ziyuglycoside I and ziyuglycoside II in Sanguisorba officinalis L. extract were 24.0 and 0.4%, respectively. Ziyuglycoside I and ziyuglycoside II standards (purity > 98.0%) were purchased from Shanghai Yansheng Technological Development (Shanghai, China), and the internal standard (IS) ginsenoside Rg3 (purity >98.0%) was purchased from Jilin University (Jilin, China). The molecular structures of ziyuglycoside I, ziyuglycoside II and IS (ginsenoside Rg3) are shown in Fig. 1. HPLC-grade acetonitrile was purchased from Merck (Merck, Germany). Deionized water was prepared by a Milli-Q system (Millipore Corporation, Billerica, MA) and was used throughout. Other reagents and solvents were commercially available and of analytical grade. 2.2 Preparation of Standards The primary stock solutions of ziyuglycoside I and ziyuglycoside II were prepared by dissolving 10.0 mg of their standards in 10 mL acetonitrile/DMSO (10:1, v/v), and were stored at 4⬚C until analysis. A mixed stock solution was prepared by mixing the primary stock solutions of ziyuglycoside I and ziyuglycoside II to yield these final concentrations: 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 g/mL. QC samples were obtained using an independent stock solution (0.05, 0.50, and 5.00 g/mL) diluted to achieve ziyuglycoside I and ziyuglycoside II concentrations of 5.0, 50.0, and 500.0 ng/mL. A set of QC samples were further stored at −20⬚C to evaluate the freeze/thaw cycle and long-term storage stability. The standards and QC samples were extracted on each analysis day with the procedures described below for plasma samples. The primary stock solutions of IS was prepared by dissolving 10.0 mg of ginsenoside Rg3 in 10 mL acetonitrile, and diluted to 0.2 g/mL using acetonitrile. 2.3 Sample preparation The plasma samples were extracted using a LLE technique. To each tube containing 100 L plasma, 10 L of IS (ginsenoside Rg3, 0.2 g/mL) and 1.0 mL of n-butanol were added. The mixture was then vortex-extracted for 2 min, and centrifuged for 10 min at 10 000 x g (4⬚C). 700 L of supernatant C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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was drawn out into Eppendorf tubes and evaporated at 45⬚C to dryness in a rotary evaporator and the residue was reconstituted in 200 L acetonitrile.
2.4 Instrument, Parameters, and Conditions The LC triple quadrupole mass spectrometer system (LC– MS/MS 8050 system, Shimadzu, Japan) was composed of a Shimadzu 30 AD LC system (Shimadzu Corporation, Japan) and an 8050 triple quadrupole mass spectrometer equipped with a heated ESI source. Data acquisition was performed using the LabSolutions LCMS Ver.5.6 software (Shimadzu, Japan). Chromatographic separation was achieved on a C18 RP Thermo Hypersil GOLD stainless-steel column (C18 , 5 m, 50 mm × 2.1 mm I.D., Thermo, USA). The aqueous mobile phase (solvent A) was as follows: H2 O containing 0.02% acetic acid, and the organic phase (solvent B) was acetonitrile. A binary gradient elution (delivered at 0.2 mL/min) was performed for the separation, and the consecutive program was as follows: an isocratic elution of 25% solvent B for the initial 0.5 min, followed by a linear gradient elution of 25– 90% solvent B from 0.5 to 4.5 min, holding the composition of 90% solvent B for the next 1.5 min followed by column equilibration to the initial conditions over 3 min. The mass spectrometer was operated in the negative mode. Quantification was obtained using selected reaction monitoring (SRM) acquisition mode by monitoring the precursor ion to product ion transitions of m/z 801.6→603.4 for ziyuglycoside I, m/z 603.6→585.4 for ziyuglycoside II and m/z 819.6→621.4 for ginsenoside Rg3 (IS). The optimized ionspray voltage and source temperature were maintained at 4000 V and 400⬚C. DL temperature was 250⬚C. The collision energy for ziyuglycoside I, ziyuglycoside II and ginsenoside Rg3 were set at 33, 38 and 39 eV, respectively. The optimized nebulizing gas was 3 L/min, and the heating gas was 10 L/min. LabSolutions LCMS Ver.5.6 software (Shimadzu, Japan) was used for the control of equipment, data acquisition and analysis.
2.5 Method validation This method was fully validated in accordance with US-FDA Bio-analytical Method Validation Guidance [16] with respect to specificity, matrix effect, linearity, sensitivity, precision, accuracy, recovery, etc. The selectivity was evaluated by comparing chromatograms of blank plasma from six different rats with the blank plasma spiked with ziyuglycoside I, ziyuglycoside II, and IS. Then the effect of the biological matrix (rat plasma) on ionization efficiency was assessed by comparing the analytical response for six extracted blank matrix samples, post-spiked with corresponding QC concentrations of compounds, with direct injected elution solution with the same nominal analyte concentration. The linearity was obtained by plotting the www.jss-journal.com
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Figure 1. The product ion mass spectra and the corresponding fragmentation pathway of ziyuglycoside I (A), ziyuglycoside II (B) and IS (C).
peak area ratios (y) of each analyte to IS against the theoretical concentrations (x) and assessed by weighed least-squares linear regression using 1/x as the weighting factor using based on LabSolutions software. In this process, blank plasma samples were also analyzed to confirm the absence of direct interferences to investigate the specificity, but these data were not used to construct the calibration curve. The lower limit of quantification (LLOQ) was defined as the lowest drug concentration that could be determined with accuracy (RE%) within ± 15% and precision (RSD%) lower than 15% based on analysis of five replicate samples. Accuracy C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is determined by replicate analysis of samples containing known amounts of the analyte, and mean value (RE) should be within ± 15% of the actual value. Precision were evaluated by five replicate assays of QC samples (5.0, 50.0 and 500.0 ng/mL) on the same day and on three consecutive days with the intra- and inter-day precision (% RSD) not exceeding 15%. The recoveries of ziyuglycoside I, ziyuglycoside II and IS were determined at three QC levels and calculated by comparing the peak areas obtained from extracted samples with those of blank matrix extracts spiked with the analytes. Stability of analytes in plasma was www.jss-journal.com
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Figure 2. Typical SRM chromatograms of (A) blank plasma, (B) blank plasma spiked with ziyuglycoside I and ziyuglycoside II at LLOQ (2.0 ng/mL) and the IS (20.0 ng/mL), (C) blank plasma spiked with 50.0 ng/ mL of ziyuglycoside I and ziyuglycoside II, and the IS (20.0 ng/mL), and (D) rat plasma collected at 2 h after intragastric administering Sanguisorba officinalis L. extract at 2.0 g/kg, and the calculated concentrations of ziyuglycoside I and ziyuglycoside II were 132.3 and 19.8 ng/mL. The retention times of ziyuglycoside I (a) ziyuglycoside II (b) and IS (c) were 3.094, 4.005, and 4.047 min, respectively. Table 1. The linearity and sensitivity of Ziyuglycoside I and Ziyuglycoside II in rat plasma (n = 5)
Ziyuglycosides
Slope (±SD)
Intercept (±SD)
Correlation coefficent
LLOQ (ng/mL)
Accuracy of LLOQ (RE, %)
Intra-day Precision of LLOQ (RSD, %)
Intra-day Precision of LLOQ (RSD, %)
I II
0.247 ± 0.012 0.075 ± 0.0044
0.067 ± 0.023 0.004 ± 0.003
0.995 0.996
2.00 2.00
–8.21 11.72
12.35 13.12
10.32 11.43
systemically investigated by analyzing three replicates of stability samples under various storage conditions: 6 h at room temperature, three freeze–thaw cycles and stored at −20⬚C for four weeks. The stability of extracted samples in autosampler for 24 h was also inspected. The analytes would be considered stable when the accuracy bias was within ± 15% of the nominal concentrations. Besides, the stock solutions of ziyuglycoside I (5.0, 50.0 and 500.0 ng/mL), ziyuglycoside II (5.0, 50.0 and 500.0 ng/mL) and IS (0.2 g/mL) were prepared in acetonitrile and stored in a fridge/freezer (4–6⬚C) for one month to investigate the stability stock solutions.
2.6 Application to a pharmacokinetic study in rats Healthy adult Sprague–Dawley rats (200 ± 20 g) were purchased from the Shanghai Super-B&K Laboratory Animal (Changsha, China) and kept in an environmentally controlled room temperature (22 ± 2⬚C) and humidity (55 ± 5⬚C) with a 12/12 h light/dark cycle. Before the experiments, rats were fed with standard food for one week to adapt to the laboratory conditions. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals (National Research Council of USA, 1996) and the related ethical regulations of our university [17]. All animals, housed on wire bottom cages, were fasted overnight (12 h) before experiments, but free access to water was available. After oral administering Sanguisorba officinalis L. extract, 200 L of blood samples were collected using heparinized tubes at 5, 10, 20, 40, 60 min, 1.5, 2, 4, 6, 8, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
12, 24, and 48 h from the ophthalmic veins and immediately centrifuged at 2000×g for 10min. The supernatant plasma was collected, and the plasma collected at 60 min was diluted twofold with blank rat plasma. Then the plasma was immediately frozen at −20⬚C until analysis.
3 Results and discussion 3.1 Selection of IS Selecting a proper IS was necessary to obtain desirable assay when mass spectrometer was as a detector. ziyuglycoside I and ziyuglycoside II both belong to triterpenoid saponins, and have similar structures with many of the common herbal saponins including ginsenosides, notoginsenosides, ophiopogonin, etc. Initially, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1(S), Rh1(R), Rh2 were all chosen as the candidates of IS. Ginsenoside Rg3 was then adopted as IS not only due to its similarity in its retention and ionization characteristics with ziyuglycoside I and ziyuglycoside II, but also because of the minimal endogenous interferences in the selected-reaction monitoring (SRM) channel for ginsenoside Rg3 (m/z 819.6→621.4). 3.2 Optimization of chromatographic and mass spectrometric conditions Conditions of chromatographic separation, especially the composition of mobile phase, played a vital role in achieving www.jss-journal.com
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Table 2. Validation results of UFLC-MS/MS on the determination of Ziyuglycoside I and Ziyuglycoside II
Nominal Concentration (ng/mL) Ziyuglycoside I Ziyuglycoside II Matrix effect (%) Ziyuglycoside I Ziyuglycoside II Accuracy (RE, %; n = 15) Ziyuglycoside I Ziyuglycoside II Intra-day Precision (RSD, %; n = 15) Ziyuglycoside I Ziyuglycoside II Inter-day Precision (RSD, %; n = 15) Ziyuglycoside I Ziyuglycoside II Recovery (%) Ziyuglycoside I Ziyuglycoside II Autosampler stability (24h; RE, %) Ziyuglycoside I Ziyuglycoside II Stability at room temperature (6h; RE, %) Ziyuglycoside I Ziyuglycoside II Freeze-thraw stability (3 cycles; RE, %) Ziyuglycoside I Ziyuglycoside II Freeze 4 weeks (–20⬚C; RE, %) Ziyuglycoside I Ziyuglycoside II
5.00 5.00
50.00 50.00
500.00 500.00
88.25 87.34
88.11 107.09
89.76 86.17
1.17 3.04
3.48 3.70
1.57 –2.41
13.34 6.01
2.71 4.82
1.86 4.56
13.93 11.47
8.73 7.86
5.37 7.86
86.80 80.21
89.69 89.83
78.05 77.45
–1.14 –1.67
6.32 3.53
4.92 7.09
1.36 –9.11
1.05 9.77
-3.07 -4.45
–4.07 2.43
7.05 7.72
1.64 1.41
–5.05 –6.41
1.15 7.53
2.49 8.46
the Q1 full scan spectra, but the intensity of chloride additive ion was much higher than the deprotonated ion. Thus, the deprotonated ions of ziyuglycoside I and ziyuglycoside II (m/z of 801.6 and 603.4) and the chloride additive ion of ginsenoside Rg3 (m/z of 819.6) were chosen as the parent ions. In addition, the collision energy was optimized from 10 to 60 eV automatically since the collision behavior was strongly dependent on the CE values. As a result, CEs at 33, 38 and 39 eV were found to be most suitable for the SRM transitions of ziyuglycoside I, ziyuglycoside II, and IS, respectively. The product ion mass spectra and their corresponding fragmentation pathway of ziyuglycoside I, ziyuglycoside II, and IS are shown in Fig. 1.
3.3 Specificity The specificity of the analytical method was determined by comparing the chromatograms of six blank rat plasma samples, plasma samples spiked with the ziyuglycoside I, ziyuglycoside II, and IS and plasma samples after a single oral dose from different individuals. Blank rat plasma samples were analyzed for endogenous interference. Representative chromatograms of blank plasma, blank plasma spiked with a standard solution at the LLOQ level, blank plasma spiked with a standard solution at 50 ng/mL, and plasma from rats following oral administration of extracts are shown in Fig. 2. Under the established chromatographic conditions, no endogenous plasma components or other impurities interferences were observed at the peak region of ziyuglycoside I (3.094 min), ziyuglycoside II (4.005 min), and IS (4.047 min).
3.4 Linearity and sensitivity good chromatographic behavior and appropriate ionization. In our experiment, several volatile additives, including ammonia water, formic acid, acetic acid, and ammonium chloride, were used for the enhancement of ionization and improvement of peak symmetry. As a result, the addition of 0.02% acetic acid to the mobile phase was found able to significantly improve the sensitivity and peak symmetry by promoting the ionization of ziyuglycoside I, ziyuglycoside II, and IS. Besides, acetonitrile was chosen as the organic phase because it could provide better peak shape and lower background noise than methanol. The full scan of ziyuglycoside I, ziyuglycoside II, and ginsenoside Rg3 in positive and negative ionization modes showed that the signals obtained in negative mode had much higher intensities than those of positive mode, and then negative ion mode was employed to investigate the analytical performance of analytes. The Q1 full scan spectra of ziyuglycoside I and ziyuglycoside II was dominant by deprotonated ion [M–H]– with m/z of 801.6 and 603.4, and chloride additive ion [M+Cl]– with m/z 837.6 and 639.5 were also observed. Similarly, deprotonated ion [M–H]– and chloride additive ion [M+Cl]– of ginsenoside Rg3 with m/z of 783.45 and 819.6 were both appeared in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The calibration curves were linear in peak area ratios over the concentration range of 2.0–1000.0 ng/mL for both ziyuglycoside I and ziyuglycoside II in rat plasma, with a correlation coefficient r > 0.99. The average calibration curves, correlation coefficients of ziyuglycoside I and ziyuglycoside II in rat plasma were y = 0.247x + 0.067 (r = 0.995) and y = 0.075x + 0.004 (r = 0.996), and the results are provided in Table 1. The LLOQs of ziyuglycoside I and ziyuglycoside II were 2.0 ng/mL with RE of 9.7 and 8.8% for ziyuglycoside I and ziyuglycoside II, respectively.
3.5 Matrix effects Matrix effect was defined as the overall effect of all components in the sample other than the analyte of interest [18]. Components originating from the sample matrix that coelute with the compound(s) of interest can cause ionization suppression or enhancement, which negatively affect the measurement of quantity of LC–MS analysis [19,20]. Herein, effects of biological matrix on ion suppression/enhancement were evaluated at three concentration levels for ziyuglycoside I/ziyuglycoside II and at a single concentration for the IS. www.jss-journal.com
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Figure 3. Mean plasma concentration–time profiles of ziyuglycoside I (A) and ziyuglycoside II (B).
Table 3. Main pharmacokinetic parameters of Ziyuglycoside I and Ziyuglycoside II after oral administration of Sanguisorba officinalis L. extract at a dose of 2.0 g/kg to rats (n = 5)
Ziyuglycoside I
Ziyuglycoside I
Parameters
Units
Mean
SD
Mean
SD
Cmax Tmax t1/2 AUC 0-t AUC 0-
(ng/mL) (h) (h) (ng.h/mL) (ng.h/mL)
723.30 0.96 19.76 4289.44 5299.09
478.89 0.39 1.59 1488.87 1258.83
88.19 2.67 12.16 1646.3 1757.97
33.35 3.56 4.44 594.56 597.67
Cmax : maximum plasma concentration; Tmax : time to reach the maximum concentration; t1/2 : elimination half time; AUC: area under concentration–time curve.
The detailed matrix results are shown in Table 2. The matrix effects were found to be within the acceptable limits (85– 115%), which indicated that ion suppression/enhancement from the rat plasma were negligible for the determination C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of ziyuglycoside I and ziyuglycoside II under the current method conditions. 3.6 Accuracy and precision The results of intra-day (n = 5) and inter-day (n = 5) accuracies and precisions of ziyuglycoside I and ziyuglycoside II estimated by evaluating three concentrations of QC samples (5.0, 50.0, 500.0 ng/mL) are shown in Table 2. Intra- and inter-day precision in rat plasma was less than 15% for all concentrations in terms of RSD, and the accuracy was below 3.48% for ziyuglycoside I and below 3.70% for ziyuglycoside II in terms of RE. All the RE (%) and RSD (%) values met the acceptance limits set by the FDA guidance Bioanalytical Method Validation (2001).
3.7 Recovery The recoveries of ziyuglycoside I, ziyuglycoside II, and IS were determined at three QC levels and calculated by www.jss-journal.com
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comparing the peak areas obtained from extracted samples with those of post-extracted samples spiked with the analytes. The recovery results are summarized in Table 2. The method provided excellent recoveries (>75%) for ziyuglycoside I and ziyuglycoside II from plasma.
3.8 Stability The stability of ziyuglycoside I and ziyuglycoside II in rat plasma under different conditions is summarized in Table 2. Excellent recoveries of ziyuglycoside I and ziyuglycoside II were observed at different storage conditions. No significant loss of ziyuglycoside I and ziyuglycoside II in rat plasma was found after storing the samples for bench-top stability at room temperature (at least 4 h), 24 h in the autosampler tray, after two weeks of storage at −20⬚C and three freeze–thaw cycles. Besides, the stability of stock solutions was determined by running the solutions stored in refrigerator for one month and the fresh made solutions, and was expressed as recovery (%) obtained by calculating the difference in peak areas. The recovery of ziyuglycoside I, ziyuglycoside II, and IS was higher than 90%, which indicated the good stability of stock solutions.
precision, accuracy, recovery, etc. Such a novel approach was successfully applied to the pharmacokinetics of ziyuglycoside I and ziyuglycoside II in rats. It is expected that our methodology will find its wide application into pharmacokinetic studies not only for ziyuglycoside I and ziyuglycoside II but for other herbal saponins as well, in view that the compounds with similar structure had similar extraction efficiency, chromatographic separation and ionization conditions. This study was supported by the National Nature Science Foundation (81374054, 81273589), the nature science foundation of Jiangsu province (BK20131311), the fundamental research special fund of China Pharmaceutical University (PT2014YK0081), Jiangsu provincial promotion foundation for the key lab of drug metabolism and pharmacokinetics (BM2012012), and Jiangsu Key laboratory of drug design and optimization. The authors have declared no conflict of interest.
5 References [1] Gao, F., Hu, Y., Fang, G., Yang, G., Xu, Z., Dou, L., Chen, Z., Fan. G., J. Pharm. Biomed. Anal. 2014, 87, 241–260.
3.9 Pharmacokinetics study
[2] Zhang, X., Guan, J., Zhu, H., Niu. T., J. Chromatogr. B. 2014, 971, 126–132.
The validated LC–ESI-MS/MS method was successfully applied to a pharmacokinetic study of ziyuglycoside I and ziyuglycoside II in rat after oral administration of Sanguisorba officinalis L. extract at a dose of 2.0 g/kg. The administered dose was chosen according to the maximum tolerated dose (MTD) and the effective dose of anti-inflammatory. Mean plasma concentration–time profiles of the two analytes are illustrated in Fig. 3. The pharmacokinetic parameters of ziyuglycoside I and ziyuglycoside II in rats were calculated by Phoenix WinNonlin pharmacokinetic program (Pharsight, Mountain View, CA) using non-compartmental analysis. The corresponding pharmacokinetic parameters including elimination half life (t1/2 ), maximum plasma concentration (Cmax ), time to reach the maximum concentrations (Tmax ) and area under concentration–time curve (AUC0-t and AUC0- ) are shown in Table 3. As a result, the Cmax , t1/2 , AUC0-t and AUC0- of ziyuglycoside I were much higher than those of ziyuglycoside II.
[3] Jung, D. Y., Seo, C. S., Kim, J. H., Shin. H. K., Int. J. Mol. Med. 2010, 26, 201–208.
4 Conclusion Ziyuglycoside I and ziyuglycoside II were the main ingredients of triterpenoid saponins exacted from Sanguisorba officinalis L., and no research about the pharmacokinetics of ziyuglycoside I and ziyuglycoside II was found until now. In the present study, a simple and sensitive LC–MS/MS method was developed and validated according to the FDA guidelines with respect to selectivity, matrix effect, linearity, sensitivity, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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[16] US Department of Health and Human Services, Food and Drug Administration. Guidance for Industry, Bioanalytical Method Validation. Center for Drug Evaluation and Research, Rockville, MD, USA (2001). [17] National Research Council of USA. Guide for the care and use of Laboratory Animals. Institute of Laboratory Animals Resources Commission on Life Sciences. National Academy Press: Washington, D.C. 1996.
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[18] Hall, T. G., Smukste, I., Bresciano, K. R., Wang, Y., McKearn, D., Savage. R. E., Rijeka, Croatia: InTech, 2012, 18. [19] Fang, N., Yu, S., Ronis, M. J., Badger, T. M., Exp. Biol. Med. (Maywood) 2014, 10. [20] Tang, D., Yu, Y., Zheng, X., Wu, J., Li, Y., Wu, X., Du, Q., Yin. X., J. Pharm. Biomed. Anal. 2014, 100, 1–10.
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Research Article
Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid–liquid extraction with n-butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C18 column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one-step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ± 15) and precision (% RSD 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II. Keywords: Liquid chromatography / Pharmacokinetics / Tandem mass spectrometry / Ziyuglycoside I / Ziyuglycoside II DOI 10.1002/jssc.201500102
1 Introduction Chinese herbal medicines had been widely used to prevent and treat diseases for thousands of years, and they are becoming more and more popular for prescription in clinical therapy for their high efficacy, ease of access, low costs, and the belief of their better compatibility with the human body and few adverse effects [1, 2]. Sanguisorba officinalis L. is a natural plant that has been traditionally used for the treatment of inflammatory and metabolic diseases, including diarrhea, chronic intestinal infections, duodenal ulcers, bleeding, etc [3–6]. A variety of chemical constituents, including triterpenoid saponins, triterpenoid flavonoids, anthraquinones, steroids, were isolated from Sanguisorba officinalis L., and pharmacological studies on their anticancer, anti-inflammatory and antioxidant properties were reported in last decade years [7–9]. Ziyuglycoside I and ziyuglycoside II are the major effective ingredients of triterpenoid saponins exacted from Sanguisorba officinalis L., and many studies have focused on Correspondence: Dr. Liang, 24 Tongjia Lane, Nanjing, Jiangsu 210009, China E-mail: [email protected] Fax: +86 25 83271060.
Abbreviations: LLOQ, lower limit of quantification; SRM, selected-reaction monitoring C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
their pharmacological activities [10–12]. In 2008, Kim et al. reported that ziyuglycoside I isolated from the S. officinalis root extract had free radical scavenging activities and elastase inhibition activities, and could be used as an active ingredient in new anti-wrinkle cosmetic products [11]. Besides, ziyuglycoside II was reported can significantly inhibit the growth of MDA-MB-435 cells through blocking cell cycle progression as well as by inducing cell apoptosis [12]. In 2012, Zhang et al. developed a LC–MS method to simultaneously quantitative analysis of seven major triterpenoids in Pyrola decorata H. Andres. However, this method was not applicable to pharmacokinetic study due to diverse matrix and different validation standard [13]. To our best knowledge, no research about the pharmacokinetic study for ziyuglycosides was reported until now. In most cases, the lack of knowledge in biological disposition was one of the major obstacles to understanding whether the Chinese herbal medicines or single compound of western medicine, and pharmacokinetic study had been proved as a crucial step to uncover the pharmacologically active substances and identify drug targets [14, 15]. Herein, a novel quantitative method for ziyuglycoside I and ziyuglycoside II was developed based on a Shimadzu
∗ Additional corresponding author: Dr. Guangji Wang E-mail: [email protected] ∗∗ These two authors contributed equally to this work.
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LC–MS/MS 8050 system, and validated considering the specificity, linearity, matrix effects, precision, accuracy, and sensitivity in accordance to the criteria established by the US Food and Drug Administration. The applicability of the method was tested using rat plasma samples obtained after intragastric administration of Sanguisorba officinalis L. extract at a dosage of 2.0 g/kg.
2 Materials and methods 2.1 Materials Sanguisorba officinalis L. extract was purchased from Nanjing Qingze Medical Technological Development (Nanjing, China), the contents of ziyuglycoside I and ziyuglycoside II in Sanguisorba officinalis L. extract were 24.0 and 0.4%, respectively. Ziyuglycoside I and ziyuglycoside II standards (purity > 98.0%) were purchased from Shanghai Yansheng Technological Development (Shanghai, China), and the internal standard (IS) ginsenoside Rg3 (purity >98.0%) was purchased from Jilin University (Jilin, China). The molecular structures of ziyuglycoside I, ziyuglycoside II and IS (ginsenoside Rg3) are shown in Fig. 1. HPLC-grade acetonitrile was purchased from Merck (Merck, Germany). Deionized water was prepared by a Milli-Q system (Millipore Corporation, Billerica, MA) and was used throughout. Other reagents and solvents were commercially available and of analytical grade. 2.2 Preparation of Standards The primary stock solutions of ziyuglycoside I and ziyuglycoside II were prepared by dissolving 10.0 mg of their standards in 10 mL acetonitrile/DMSO (10:1, v/v), and were stored at 4⬚C until analysis. A mixed stock solution was prepared by mixing the primary stock solutions of ziyuglycoside I and ziyuglycoside II to yield these final concentrations: 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 g/mL. QC samples were obtained using an independent stock solution (0.05, 0.50, and 5.00 g/mL) diluted to achieve ziyuglycoside I and ziyuglycoside II concentrations of 5.0, 50.0, and 500.0 ng/mL. A set of QC samples were further stored at −20⬚C to evaluate the freeze/thaw cycle and long-term storage stability. The standards and QC samples were extracted on each analysis day with the procedures described below for plasma samples. The primary stock solutions of IS was prepared by dissolving 10.0 mg of ginsenoside Rg3 in 10 mL acetonitrile, and diluted to 0.2 g/mL using acetonitrile. 2.3 Sample preparation The plasma samples were extracted using a LLE technique. To each tube containing 100 L plasma, 10 L of IS (ginsenoside Rg3, 0.2 g/mL) and 1.0 mL of n-butanol were added. The mixture was then vortex-extracted for 2 min, and centrifuged for 10 min at 10 000 x g (4⬚C). 700 L of supernatant C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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was drawn out into Eppendorf tubes and evaporated at 45⬚C to dryness in a rotary evaporator and the residue was reconstituted in 200 L acetonitrile.
2.4 Instrument, Parameters, and Conditions The LC triple quadrupole mass spectrometer system (LC– MS/MS 8050 system, Shimadzu, Japan) was composed of a Shimadzu 30 AD LC system (Shimadzu Corporation, Japan) and an 8050 triple quadrupole mass spectrometer equipped with a heated ESI source. Data acquisition was performed using the LabSolutions LCMS Ver.5.6 software (Shimadzu, Japan). Chromatographic separation was achieved on a C18 RP Thermo Hypersil GOLD stainless-steel column (C18 , 5 m, 50 mm × 2.1 mm I.D., Thermo, USA). The aqueous mobile phase (solvent A) was as follows: H2 O containing 0.02% acetic acid, and the organic phase (solvent B) was acetonitrile. A binary gradient elution (delivered at 0.2 mL/min) was performed for the separation, and the consecutive program was as follows: an isocratic elution of 25% solvent B for the initial 0.5 min, followed by a linear gradient elution of 25– 90% solvent B from 0.5 to 4.5 min, holding the composition of 90% solvent B for the next 1.5 min followed by column equilibration to the initial conditions over 3 min. The mass spectrometer was operated in the negative mode. Quantification was obtained using selected reaction monitoring (SRM) acquisition mode by monitoring the precursor ion to product ion transitions of m/z 801.6→603.4 for ziyuglycoside I, m/z 603.6→585.4 for ziyuglycoside II and m/z 819.6→621.4 for ginsenoside Rg3 (IS). The optimized ionspray voltage and source temperature were maintained at 4000 V and 400⬚C. DL temperature was 250⬚C. The collision energy for ziyuglycoside I, ziyuglycoside II and ginsenoside Rg3 were set at 33, 38 and 39 eV, respectively. The optimized nebulizing gas was 3 L/min, and the heating gas was 10 L/min. LabSolutions LCMS Ver.5.6 software (Shimadzu, Japan) was used for the control of equipment, data acquisition and analysis.
2.5 Method validation This method was fully validated in accordance with US-FDA Bio-analytical Method Validation Guidance [16] with respect to specificity, matrix effect, linearity, sensitivity, precision, accuracy, recovery, etc. The selectivity was evaluated by comparing chromatograms of blank plasma from six different rats with the blank plasma spiked with ziyuglycoside I, ziyuglycoside II, and IS. Then the effect of the biological matrix (rat plasma) on ionization efficiency was assessed by comparing the analytical response for six extracted blank matrix samples, post-spiked with corresponding QC concentrations of compounds, with direct injected elution solution with the same nominal analyte concentration. The linearity was obtained by plotting the www.jss-journal.com
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Figure 1. The product ion mass spectra and the corresponding fragmentation pathway of ziyuglycoside I (A), ziyuglycoside II (B) and IS (C).
peak area ratios (y) of each analyte to IS against the theoretical concentrations (x) and assessed by weighed least-squares linear regression using 1/x as the weighting factor using based on LabSolutions software. In this process, blank plasma samples were also analyzed to confirm the absence of direct interferences to investigate the specificity, but these data were not used to construct the calibration curve. The lower limit of quantification (LLOQ) was defined as the lowest drug concentration that could be determined with accuracy (RE%) within ± 15% and precision (RSD%) lower than 15% based on analysis of five replicate samples. Accuracy C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is determined by replicate analysis of samples containing known amounts of the analyte, and mean value (RE) should be within ± 15% of the actual value. Precision were evaluated by five replicate assays of QC samples (5.0, 50.0 and 500.0 ng/mL) on the same day and on three consecutive days with the intra- and inter-day precision (% RSD) not exceeding 15%. The recoveries of ziyuglycoside I, ziyuglycoside II and IS were determined at three QC levels and calculated by comparing the peak areas obtained from extracted samples with those of blank matrix extracts spiked with the analytes. Stability of analytes in plasma was www.jss-journal.com
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Figure 2. Typical SRM chromatograms of (A) blank plasma, (B) blank plasma spiked with ziyuglycoside I and ziyuglycoside II at LLOQ (2.0 ng/mL) and the IS (20.0 ng/mL), (C) blank plasma spiked with 50.0 ng/ mL of ziyuglycoside I and ziyuglycoside II, and the IS (20.0 ng/mL), and (D) rat plasma collected at 2 h after intragastric administering Sanguisorba officinalis L. extract at 2.0 g/kg, and the calculated concentrations of ziyuglycoside I and ziyuglycoside II were 132.3 and 19.8 ng/mL. The retention times of ziyuglycoside I (a) ziyuglycoside II (b) and IS (c) were 3.094, 4.005, and 4.047 min, respectively. Table 1. The linearity and sensitivity of Ziyuglycoside I and Ziyuglycoside II in rat plasma (n = 5)
Ziyuglycosides
Slope (±SD)
Intercept (±SD)
Correlation coefficent
LLOQ (ng/mL)
Accuracy of LLOQ (RE, %)
Intra-day Precision of LLOQ (RSD, %)
Intra-day Precision of LLOQ (RSD, %)
I II
0.247 ± 0.012 0.075 ± 0.0044
0.067 ± 0.023 0.004 ± 0.003
0.995 0.996
2.00 2.00
–8.21 11.72
12.35 13.12
10.32 11.43
systemically investigated by analyzing three replicates of stability samples under various storage conditions: 6 h at room temperature, three freeze–thaw cycles and stored at −20⬚C for four weeks. The stability of extracted samples in autosampler for 24 h was also inspected. The analytes would be considered stable when the accuracy bias was within ± 15% of the nominal concentrations. Besides, the stock solutions of ziyuglycoside I (5.0, 50.0 and 500.0 ng/mL), ziyuglycoside II (5.0, 50.0 and 500.0 ng/mL) and IS (0.2 g/mL) were prepared in acetonitrile and stored in a fridge/freezer (4–6⬚C) for one month to investigate the stability stock solutions.
2.6 Application to a pharmacokinetic study in rats Healthy adult Sprague–Dawley rats (200 ± 20 g) were purchased from the Shanghai Super-B&K Laboratory Animal (Changsha, China) and kept in an environmentally controlled room temperature (22 ± 2⬚C) and humidity (55 ± 5⬚C) with a 12/12 h light/dark cycle. Before the experiments, rats were fed with standard food for one week to adapt to the laboratory conditions. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals (National Research Council of USA, 1996) and the related ethical regulations of our university [17]. All animals, housed on wire bottom cages, were fasted overnight (12 h) before experiments, but free access to water was available. After oral administering Sanguisorba officinalis L. extract, 200 L of blood samples were collected using heparinized tubes at 5, 10, 20, 40, 60 min, 1.5, 2, 4, 6, 8, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
12, 24, and 48 h from the ophthalmic veins and immediately centrifuged at 2000×g for 10min. The supernatant plasma was collected, and the plasma collected at 60 min was diluted twofold with blank rat plasma. Then the plasma was immediately frozen at −20⬚C until analysis.
3 Results and discussion 3.1 Selection of IS Selecting a proper IS was necessary to obtain desirable assay when mass spectrometer was as a detector. ziyuglycoside I and ziyuglycoside II both belong to triterpenoid saponins, and have similar structures with many of the common herbal saponins including ginsenosides, notoginsenosides, ophiopogonin, etc. Initially, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1(S), Rh1(R), Rh2 were all chosen as the candidates of IS. Ginsenoside Rg3 was then adopted as IS not only due to its similarity in its retention and ionization characteristics with ziyuglycoside I and ziyuglycoside II, but also because of the minimal endogenous interferences in the selected-reaction monitoring (SRM) channel for ginsenoside Rg3 (m/z 819.6→621.4). 3.2 Optimization of chromatographic and mass spectrometric conditions Conditions of chromatographic separation, especially the composition of mobile phase, played a vital role in achieving www.jss-journal.com
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Table 2. Validation results of UFLC-MS/MS on the determination of Ziyuglycoside I and Ziyuglycoside II
Nominal Concentration (ng/mL) Ziyuglycoside I Ziyuglycoside II Matrix effect (%) Ziyuglycoside I Ziyuglycoside II Accuracy (RE, %; n = 15) Ziyuglycoside I Ziyuglycoside II Intra-day Precision (RSD, %; n = 15) Ziyuglycoside I Ziyuglycoside II Inter-day Precision (RSD, %; n = 15) Ziyuglycoside I Ziyuglycoside II Recovery (%) Ziyuglycoside I Ziyuglycoside II Autosampler stability (24h; RE, %) Ziyuglycoside I Ziyuglycoside II Stability at room temperature (6h; RE, %) Ziyuglycoside I Ziyuglycoside II Freeze-thraw stability (3 cycles; RE, %) Ziyuglycoside I Ziyuglycoside II Freeze 4 weeks (–20⬚C; RE, %) Ziyuglycoside I Ziyuglycoside II
5.00 5.00
50.00 50.00
500.00 500.00
88.25 87.34
88.11 107.09
89.76 86.17
1.17 3.04
3.48 3.70
1.57 –2.41
13.34 6.01
2.71 4.82
1.86 4.56
13.93 11.47
8.73 7.86
5.37 7.86
86.80 80.21
89.69 89.83
78.05 77.45
–1.14 –1.67
6.32 3.53
4.92 7.09
1.36 –9.11
1.05 9.77
-3.07 -4.45
–4.07 2.43
7.05 7.72
1.64 1.41
–5.05 –6.41
1.15 7.53
2.49 8.46
the Q1 full scan spectra, but the intensity of chloride additive ion was much higher than the deprotonated ion. Thus, the deprotonated ions of ziyuglycoside I and ziyuglycoside II (m/z of 801.6 and 603.4) and the chloride additive ion of ginsenoside Rg3 (m/z of 819.6) were chosen as the parent ions. In addition, the collision energy was optimized from 10 to 60 eV automatically since the collision behavior was strongly dependent on the CE values. As a result, CEs at 33, 38 and 39 eV were found to be most suitable for the SRM transitions of ziyuglycoside I, ziyuglycoside II, and IS, respectively. The product ion mass spectra and their corresponding fragmentation pathway of ziyuglycoside I, ziyuglycoside II, and IS are shown in Fig. 1.
3.3 Specificity The specificity of the analytical method was determined by comparing the chromatograms of six blank rat plasma samples, plasma samples spiked with the ziyuglycoside I, ziyuglycoside II, and IS and plasma samples after a single oral dose from different individuals. Blank rat plasma samples were analyzed for endogenous interference. Representative chromatograms of blank plasma, blank plasma spiked with a standard solution at the LLOQ level, blank plasma spiked with a standard solution at 50 ng/mL, and plasma from rats following oral administration of extracts are shown in Fig. 2. Under the established chromatographic conditions, no endogenous plasma components or other impurities interferences were observed at the peak region of ziyuglycoside I (3.094 min), ziyuglycoside II (4.005 min), and IS (4.047 min).
3.4 Linearity and sensitivity good chromatographic behavior and appropriate ionization. In our experiment, several volatile additives, including ammonia water, formic acid, acetic acid, and ammonium chloride, were used for the enhancement of ionization and improvement of peak symmetry. As a result, the addition of 0.02% acetic acid to the mobile phase was found able to significantly improve the sensitivity and peak symmetry by promoting the ionization of ziyuglycoside I, ziyuglycoside II, and IS. Besides, acetonitrile was chosen as the organic phase because it could provide better peak shape and lower background noise than methanol. The full scan of ziyuglycoside I, ziyuglycoside II, and ginsenoside Rg3 in positive and negative ionization modes showed that the signals obtained in negative mode had much higher intensities than those of positive mode, and then negative ion mode was employed to investigate the analytical performance of analytes. The Q1 full scan spectra of ziyuglycoside I and ziyuglycoside II was dominant by deprotonated ion [M–H]– with m/z of 801.6 and 603.4, and chloride additive ion [M+Cl]– with m/z 837.6 and 639.5 were also observed. Similarly, deprotonated ion [M–H]– and chloride additive ion [M+Cl]– of ginsenoside Rg3 with m/z of 783.45 and 819.6 were both appeared in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The calibration curves were linear in peak area ratios over the concentration range of 2.0–1000.0 ng/mL for both ziyuglycoside I and ziyuglycoside II in rat plasma, with a correlation coefficient r > 0.99. The average calibration curves, correlation coefficients of ziyuglycoside I and ziyuglycoside II in rat plasma were y = 0.247x + 0.067 (r = 0.995) and y = 0.075x + 0.004 (r = 0.996), and the results are provided in Table 1. The LLOQs of ziyuglycoside I and ziyuglycoside II were 2.0 ng/mL with RE of 9.7 and 8.8% for ziyuglycoside I and ziyuglycoside II, respectively.
3.5 Matrix effects Matrix effect was defined as the overall effect of all components in the sample other than the analyte of interest [18]. Components originating from the sample matrix that coelute with the compound(s) of interest can cause ionization suppression or enhancement, which negatively affect the measurement of quantity of LC–MS analysis [19,20]. Herein, effects of biological matrix on ion suppression/enhancement were evaluated at three concentration levels for ziyuglycoside I/ziyuglycoside II and at a single concentration for the IS. www.jss-journal.com
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Figure 3. Mean plasma concentration–time profiles of ziyuglycoside I (A) and ziyuglycoside II (B).
Table 3. Main pharmacokinetic parameters of Ziyuglycoside I and Ziyuglycoside II after oral administration of Sanguisorba officinalis L. extract at a dose of 2.0 g/kg to rats (n = 5)
Ziyuglycoside I
Ziyuglycoside I
Parameters
Units
Mean
SD
Mean
SD
Cmax Tmax t1/2 AUC 0-t AUC 0-
(ng/mL) (h) (h) (ng.h/mL) (ng.h/mL)
723.30 0.96 19.76 4289.44 5299.09
478.89 0.39 1.59 1488.87 1258.83
88.19 2.67 12.16 1646.3 1757.97
33.35 3.56 4.44 594.56 597.67
Cmax : maximum plasma concentration; Tmax : time to reach the maximum concentration; t1/2 : elimination half time; AUC: area under concentration–time curve.
The detailed matrix results are shown in Table 2. The matrix effects were found to be within the acceptable limits (85– 115%), which indicated that ion suppression/enhancement from the rat plasma were negligible for the determination C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of ziyuglycoside I and ziyuglycoside II under the current method conditions. 3.6 Accuracy and precision The results of intra-day (n = 5) and inter-day (n = 5) accuracies and precisions of ziyuglycoside I and ziyuglycoside II estimated by evaluating three concentrations of QC samples (5.0, 50.0, 500.0 ng/mL) are shown in Table 2. Intra- and inter-day precision in rat plasma was less than 15% for all concentrations in terms of RSD, and the accuracy was below 3.48% for ziyuglycoside I and below 3.70% for ziyuglycoside II in terms of RE. All the RE (%) and RSD (%) values met the acceptance limits set by the FDA guidance Bioanalytical Method Validation (2001).
3.7 Recovery The recoveries of ziyuglycoside I, ziyuglycoside II, and IS were determined at three QC levels and calculated by www.jss-journal.com
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comparing the peak areas obtained from extracted samples with those of post-extracted samples spiked with the analytes. The recovery results are summarized in Table 2. The method provided excellent recoveries (>75%) for ziyuglycoside I and ziyuglycoside II from plasma.
3.8 Stability The stability of ziyuglycoside I and ziyuglycoside II in rat plasma under different conditions is summarized in Table 2. Excellent recoveries of ziyuglycoside I and ziyuglycoside II were observed at different storage conditions. No significant loss of ziyuglycoside I and ziyuglycoside II in rat plasma was found after storing the samples for bench-top stability at room temperature (at least 4 h), 24 h in the autosampler tray, after two weeks of storage at −20⬚C and three freeze–thaw cycles. Besides, the stability of stock solutions was determined by running the solutions stored in refrigerator for one month and the fresh made solutions, and was expressed as recovery (%) obtained by calculating the difference in peak areas. The recovery of ziyuglycoside I, ziyuglycoside II, and IS was higher than 90%, which indicated the good stability of stock solutions.
precision, accuracy, recovery, etc. Such a novel approach was successfully applied to the pharmacokinetics of ziyuglycoside I and ziyuglycoside II in rats. It is expected that our methodology will find its wide application into pharmacokinetic studies not only for ziyuglycoside I and ziyuglycoside II but for other herbal saponins as well, in view that the compounds with similar structure had similar extraction efficiency, chromatographic separation and ionization conditions. This study was supported by the National Nature Science Foundation (81374054, 81273589), the nature science foundation of Jiangsu province (BK20131311), the fundamental research special fund of China Pharmaceutical University (PT2014YK0081), Jiangsu provincial promotion foundation for the key lab of drug metabolism and pharmacokinetics (BM2012012), and Jiangsu Key laboratory of drug design and optimization. The authors have declared no conflict of interest.
5 References [1] Gao, F., Hu, Y., Fang, G., Yang, G., Xu, Z., Dou, L., Chen, Z., Fan. G., J. Pharm. Biomed. Anal. 2014, 87, 241–260.
3.9 Pharmacokinetics study
[2] Zhang, X., Guan, J., Zhu, H., Niu. T., J. Chromatogr. B. 2014, 971, 126–132.
The validated LC–ESI-MS/MS method was successfully applied to a pharmacokinetic study of ziyuglycoside I and ziyuglycoside II in rat after oral administration of Sanguisorba officinalis L. extract at a dose of 2.0 g/kg. The administered dose was chosen according to the maximum tolerated dose (MTD) and the effective dose of anti-inflammatory. Mean plasma concentration–time profiles of the two analytes are illustrated in Fig. 3. The pharmacokinetic parameters of ziyuglycoside I and ziyuglycoside II in rats were calculated by Phoenix WinNonlin pharmacokinetic program (Pharsight, Mountain View, CA) using non-compartmental analysis. The corresponding pharmacokinetic parameters including elimination half life (t1/2 ), maximum plasma concentration (Cmax ), time to reach the maximum concentrations (Tmax ) and area under concentration–time curve (AUC0-t and AUC0- ) are shown in Table 3. As a result, the Cmax , t1/2 , AUC0-t and AUC0- of ziyuglycoside I were much higher than those of ziyuglycoside II.
[3] Jung, D. Y., Seo, C. S., Kim, J. H., Shin. H. K., Int. J. Mol. Med. 2010, 26, 201–208.
4 Conclusion Ziyuglycoside I and ziyuglycoside II were the main ingredients of triterpenoid saponins exacted from Sanguisorba officinalis L., and no research about the pharmacokinetics of ziyuglycoside I and ziyuglycoside II was found until now. In the present study, a simple and sensitive LC–MS/MS method was developed and validated according to the FDA guidelines with respect to selectivity, matrix effect, linearity, sensitivity, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[4] Cai, Z., Li, W., Wang, H., Yan, W., Zhou, Y., Wang, G., Cui, J., Wang, F., Int. J. Biol. Macromol. 2012, 51, 484–488. [5] Wang, Z., Loo, W. T., Wang, N., Chow, L.W., Wang, D., Han, F., Zheng, X., Chen. J. P., Expert Opin. Ther. Targets, 2012, 16, S79L 89. [6] Choi, E. S., Kim, J. S., Kwon, K. H., Kim, H. S., Cho, N. P., Cho. S. D., Mol Med Rep. 2012, 6, 670–674. [7] Sun, W., Zhang, Z. L., Liu, X., Zhang, S., He, L., Wang, Z., Wang. G. S., Molecules. 2012, 17, 7629–7636. [8] Yu, B. B., Zhong, F. X., Dong. X., Chin. J. Inf. TCM. 2009, 16, 103–105. [9] Xia, H. M., Sun, L. L., Sun, J. Y., Zhong, Y., Food Drug. 2009, 11, 67–69. [10] Zhu, A. K., Zhou, H., Xia, J. Z., Jin, H. C., Wang, K., Yan, J., Zuo, J. B., Zhu, X., Shan, T., Braz. J. Med. Biol. Res. 2013, 46, 670–675. [11] Kim, Y. H., Chung, C. B., Kim, J. G., Ko, K. I., Park, S. H., Kim, J. H., Eom, S. Y., Kim, Y. S., Hwang, Y. I., Kim. K. H., Biosci. Biotechnol. Biochem. 2008, 72, 303–311. [12] Zhu, X., Wang, K., Zhang, K., Huang, B., Zhang, J., Zhang, Y., Zhu, L., Zhou, B., Zhou, F., Int. J. Mol. Sci. 2013, 14, 18041–18055. [13] Zhang, Y. Y., Zhang, C., Ren, R., Liu, R., Pharmazie. 2012, 67, 822–826. [14] Gong, P., Cui, N., Wu, L., Liang, Y., Hao, K., Xu, X., Tang, W., Wang, G., Hao. H., Anal. Chem. 2012, 84, 2995–3002. [15] Hao, H., Zheng, X., Wang. G., Trends Pharmacol. Sci. 2014, 35, 168–177.
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[16] US Department of Health and Human Services, Food and Drug Administration. Guidance for Industry, Bioanalytical Method Validation. Center for Drug Evaluation and Research, Rockville, MD, USA (2001). [17] National Research Council of USA. Guide for the care and use of Laboratory Animals. Institute of Laboratory Animals Resources Commission on Life Sciences. National Academy Press: Washington, D.C. 1996.
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[18] Hall, T. G., Smukste, I., Bresciano, K. R., Wang, Y., McKearn, D., Savage. R. E., Rijeka, Croatia: InTech, 2012, 18. [19] Fang, N., Yu, S., Ronis, M. J., Badger, T. M., Exp. Biol. Med. (Maywood) 2014, 10. [20] Tang, D., Yu, Y., Zheng, X., Wu, J., Li, Y., Wu, X., Du, Q., Yin. X., J. Pharm. Biomed. Anal. 2014, 100, 1–10.
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